As recombinant DNA technology has developed in recent years, the controlled production by microorganisms of an enormous variety of useful polypeptides has become possible. Many eukaryotic polypeptides, such as for example, human growth hormone, leukocyte interferons, human insulin and human proinsulin have already been produced by various microorganisms. The continued application of techniques already in hand is expected in the future to permit production by microorganisms of a variety of other useful polypeptide products.
A basic element frequently employed in recombinant technology is the plasmid, which is extrachromosomal, double-stranded DNA found in some microorganisms. Where plasmids have been found to naturally occur in microorganisms, they are often found to occur in multiple copies per cell. In addition to naturally occurring plasmids, a variety of man-made plasmids, or hybrid vectors, have been prepared. Unfortunately, it is not always possible for the host cell to maintain the plasmid. Instead, the plasmid is lost as the organism reproduces and passes through several generations of growth. Methods for the stable introduction of foreign DNA into suitable host organisms are therefore of great interest and potentially of great value.
Up to now, commercial efforts employing recombinant DNA technology for producing various polypeptides have centered on Escherichia coli as a host organism. However, in some situations E. coli may prove to be unsuitable as a host. For example, E. coli contains a number of toxic pyrogenic factors that must be eliminated from any polypeptide useful as a pharmaceutical product. The efficiency with which this purification can be achieved will, of course, vary with the particular polypeptide. In addition, the proteolytic activities of E. coli can seriously limit yields of some useful products. Furthermore, a number of heterologous gene products which have been produced in E. coli have been found to be produced in insoluble form. These and other considerations have led to increased interest in alternate hosts. In particular, the use of eukaryotic organisms for the production of polypeptide products is appealing.
The availability of means for the production of polypeptide products in eukaryotic systems, e.g., yeast, could provide significant advantages relative to the use of prokaryotic systems such as E. coli for the production of polypeptides encoded by recombinant DNA. Yeast has been employed in large scale fermentation for centuries, as compared to the relatively recent advent of large scale E. coli fermentations. Yeast can generally be grown to higher cell densities than bacteria and are readily adaptable to continuous fermentation processing. In fact, growth of yeast such as Pichia pastoris to ultra-high cell densities, i.e., cell densities in excess of 100 g/L, is disclosed by Wegner in U.S. Pat. No. 4,414,329 (assigned to Phillips Petroleum Co.). Additional advantages of yeast hosts include the fact that many critical functions of the organism, e.g., oxidative phosphorylation, are located within organelles, and hence are not exposed to possible deleterious effects caused by the organisms production of polypeptides foreign to the wild-type host cells. As a eukaryotic organism, yeast may prove capable of glycosylating expressed polypeptide products, which may prove of value where such glycosylation is important to the bioactivity of the polypeptide product. It is also possible that as a eukaryotic organism, yeast will exhibit the same codon preferences as higher organisms, thus tending toward more efficient production of expression products from mammalian genes or from complementary DNA (cDNA) obtained by reverse transciption from, for example, mammalian mRNA.
The development of poorly characterized yeast species as host/vector systems is severely hampered by the lack of knowledge about transformation conditions and suitable means for stably introducing foreign DNA into the host cell. In addition, auxotrophic mutants are often not available, precluding a direct selection for transformants by auxotrophic complementation. If recombinant DNA technology is to fully sustain its promise, new host/vector systems must be devised which facilitate the manipulation of DNA as well as optimize the expression of inserted DNA sequences so that the desired polypeptide products can be prepared under controlled conditions and in high yield.